Phosphatidylinositol-3,5-bisphosphate (PtdIns(3,5)P2) is one of the most recently identified phosphoinositides, and plays important roles in trafficking, protein sorting, and stress responses in mammalian and yeast cells. A family of beta-propeller proteins, exemplified by the yeast protein Svp1p, has recently been identified as high-affinity and specific binding proteins (and effectors) for PtdIns(3,5)P2. Svp1p and its relatives share no similarity with other phosphoinositide binding proteins. In this application I propose studies aimed at understanding how these proteins function as PtdIns(3,5)P2 effectors. My specific aims are : 1. To test the hypothesis that all Svp1p-related proteins specifically recognize PtdIns(3,5)P2 in vitro and in vivo - i.e. that they do not represent a family of general phosphoinositide-binding proteins. 2. To determine the mechanism of PtdIns(3,5)P2 recognition and binding by Svp1p and its relatives, as well as the stoichiometry and thermodynamic characteristics. 3. To determine a high-resolution crystal structure of Svp1p and its closest human homolog (hSvp1a) both alone and in complex with the PtdIns(3,5)P2 headgroup. These studies will define a new family of proteins with a specific phosphoinositide target, and allow further insight into how they function as effectors to mediate the plethora of cellular effects ascribed to PtdIns(3,5)P2.